Early embryonic blood vessels form with great anatomical reproducibility to ensure homeostasis and survival. How the vasculature acquires its stereotypical architecture is poorly understood. To uncover the genetic and cellular basis of vascular patterning we study the development of the zebrafish Segmental (Se) vessels. These vessels have a simple pattern, are made of few endothelial cells whose behaviors are easily observed and can be studied with genetic, chemical and embryological tools. We have shown that paracrine, repellent Semaphorin (Sema) cues sensed by the endothelial-specific PlexinD1 (PlxnD1) receptor shape the Se vessel anatomy. What molecular events occur inside the endothelial cell during Sema-PlxnD1 signaling? The mechanistic basis of intracellular PlxnD1 function and the molecular players involved are unknown. PlxnD1 has cytosolic domains similar to those essential in axonal Plxns for repulsive activity and also PlxnD1- specific sequence features. Whether these motifs are required for PlxnD1's vascular patterning activity remains unexplored. Thus, in Specific Aim 1 we will ask if these domains are required for PlxnD1-mediated Se vessel patterning and define their cellular and biochemical roles. Next, we will characterize gipc1, the first identified candidate modulator/effector of PlxnD1 signaling. gipc1 is specifically transcribed in the developing vessels, it encodes a protein that physically associates with the PlxnD1 cytosolic tail and its activity is essential for Se vessel patterning. Thus, in Specific Aim 2 we will determine the molecular and cellular roles of GIPC1 for PlxnD1-mediated Se vessel patterning. To do this we will define the GIPC1 domains that mediate its physical association with PlxnD1, ask if this interaction is necessary for PlxnD1-mediated Se vessel patterning, define if GIPC1 promotes or antagonizes PlxnD1 signaling and analyze the Se vessel patterning phenotypes of animals with reduced or increased levels of gipc1. Finally, in Specific Aim 3 we focus on road to perdition (rtp), a mutant with Se vessel defects similar to those of plxnD1 nulls. We will start by cloning rtp to explore its relationship to PlxnD1 signaling. Then, we will determine the endothelial cell behaviors that are rtp-dependent. Our studies promise to offer key insights into the genetic programs and cellular behaviors that shape the vascular tree in both health and disease. PUBLIC HEALTH RELEVANCE: The reproducible anatomical organization of the vaculature is essential for homeostasis and survival. PlexinD1 is an endothelial specific receptor that plays a key role in shaping the vascular pattern. In this proposal we address the molecular and cellular mechanism of vascular patterning by PlexinD1 signaling using zebrafish.